1.1 Field of the Invention
The present invention relates generally to the fields of oncology and molecular biology. More particularly, it concerns nucleic acid segments isolated from human chromosome 1p31, which encode a novel tumor suppressor protein, designated NOEY2. Various methods for making and using NOEY2 DNA segments, DNA segments encoding synthetically-modified NOEY2 proteins, and native and synthetic tumor suppressor proteins are disclosed, such as, for example, the use of DNA segments as diagnostic probes and templates for protein production, and the use of proteins, fusion protein carriers and peptides in various immunological and diagnostic applications. Also disclosed are methods for identifying NOEY2-related tumor suppressor polynucleotides and polypeptides, and methods for treating tumors, and in particular, ovarian and breast-related cancers.
1.2 Description of Related Art
Oncogenesis was described by Foulds (1958) as a multistep biological process, which is presently known to occur by the accumulation of genetic damage. On a molecular level, the multistep process of tumorigenesis involves the disruption of both positive and negative regulatory effectors (Weinberg, 1989). Defects leading to the development of retinoblastoma have been linked to a tumor suppressor gene (Lee et al., 1987), and a variety of oncogenes and other tumor suppressors have been identified in a host of malignancies. Unfortunately, there remains an inadequate number of treatable cancers, and the effects of cancer are catastrophic—over half a million deaths per year in the United States alone.
1.2.1 Tumor Suppressors
Tumor suppressor proteins function to negatively regulate cell cycle processes, preventing the uncontrolled growth exhibited by cancerous cells. Tumor suppressor proteins function by transcriptional regulation of key genes involved in cellular growth and division. Unlike protooncogenes, for which activation is required for the initiation of cancerous growth, inactivation of tumor suppressors leads to cancer. In normal diploid cells, two copies of the gene encoding a particular tumor suppressor protein are present. The “two hit” hypothesis states that mutation or inactivation of both individual copies of the gene is required for the onset of cancerous growth. Mutations may include deletions, alterations to transcription levels, single or multiple coding changes, and truncations. Inherited mutations in a single tumor suppressor gene typically leads to a recessive heterozygous phenotype. Cells remain phenotypically normal until mutation of the second, wild type copy of the gene. Familial inheritance of a mutant copy of a tumor suppressor gene does render the individual more prone to cancer, as inactivation of the single normal gene is sufficient to initiate cancer. The two most heavily studied tumor suppressor genes are the retinoblastoma gene Rb and the p53 gene.
The retinoblastoma gene Rb was the first discovered tumor suppressor gene. The Rb protein is a 110 kDa nuclear phosphoprotein that reduces the growth rate of cells, and mutations in this protein have been found in breast, prostate, and small cell lung carcinomas. The Rb protein binds to DNA, but without any sequence specificity. Rb effects its regulatory function through the formation of complexes with transcription factors. Rb has been shown to interact with transcription factor E2F, which binds to the promoters of cellular genes such as DNA polymerase α and ribonucleotide reductase. Heterozygous individuals inherit one defective and one normal copy of the Rb gene. Upon inactivation of the second, functional copy of the gene, cancerous growth initiates. A wide range of mutations including deletions, duplications, and point mutations have been found to lead to inactivation of the Rb protein.
The p53 tumor suppressor has been found to be mutated in about 60% of human cancerous growths, making p53 the most commonly mutated gene in human cancers. Wild type p53 protein binds to DNA and is believed to function as a transcriptional regulator. The p53 protein also binds to the mdm2 protein, commonly expressed at high levels in tumors. Levels of wild type p53 protein increases upon subjecting a cell to radiation or chemical agents which damage DNA. The p53 protein has been implicated in DNA repair mechanisms that prevent the duplication of damaged or altered DNA. It has been further speculated that p53 protein prevents the cell from entering S phase until the complete repair of damaged DNA has been achieved. The p53 protein is normally found at low, often undetectable levels in cells, and has a short cellular half life. Mutant p53 proteins often are more resistant to degradation, and are present at immunohistochemically detectable levels in cancerous cells. Most p53 gene mutations occur in four highly conserved regions. Mutant p53 proteins are dominant inactivators, by their ability to bind to and inactivate wild type p53 protein.
1.2.2 Ovarian Cancer
The ovary is the fifth most common site of cancer among American women and ovarian neoplasms constitute the fourth leading cause of cancer death. Ovarian cancer affects 22,000 women in the United States each year and causes some 14,000 deaths annually. Approximately 90% of ovarian cancers arise from epithelial cells that cover the ovarian surface or that line inclusion cysts. Ovarian cancers exhibit a distinctive pattern of metastasis. Like other epithelial neoplasms, ovarian cancer can metastasize to pelvic and retroperitoneal lymph nodes and can spread hematogenously to distant sites. More frequently, however, ovarian cancer cells spread over the surface of the peritoneal cavity, forming multiple nodules on the parietal and visceral peritoneum. Blockade of diaphragmatic lymphatics and increased transudation of fluid produce accumulation of ascites fluid that contains varying numbers of tumor cells. Abdominal distention by malignant ascites is a frequent mode of clinical presentation. Early stage ovarian cancer rarely produces symptoms and at present there is no proven strategy for early detection. In more than 75% of cases, tumor cells have metastasized beyond the ovaries at presentation. Initial clinical management generally involves cytoreductive surgery and drainage of ascites, providing large amounts of tissue for study. Following removal of as much tumor as possible, cytotoxic chemotherapy is generally administered. Introduction of platinum based compounds and the taxane derivatives has improved median survival of patients with advanced disease, but the five year survival rate is still only 28% for all stages and has not improved in the last several decades. Disease frequently persists and recurs within the peritoneal cavity, producing intestinal obstruction. Therapeutic strategies that prevent such progression and that treat disease regionally by intraperitoneal infusion continue to hold promise. Identification of genes whose aberrant function can be demonstrated in ovarian carcinomas may have important diagnostic and therapeutic applications.
1.2.3 Breast Cancer
Breast cancer is the most common form of cancer among women, affecting about one in eight women. Approximately 185,700 new cases are diagnosed in the U.S. annually, and breast cancer is responsible for about 44,560 deaths in the U.S. per year. While predominantly observed in women, 1,400 cases of breast cancer are diagnosed annually in men, and 260 men die of breast cancer per year. Breast cancer first manifests itself as a painless lump, detectable by self-examination and clinical breast exams including mammograms. Commonly, growth initiates in the lining of the ducts or in the lobules of the breast. Current clinical treatments include mastectomy (removal of the entire breast) or lumpectomy (removal of the tumor and surrounding tissue) for localized tumors. Chemotherapy, radiotherapy, or hormone-blocking therapy may be further used to control cancerous cells. Breast cancer cells can metastasize to the lymph nodes, skin, lungs, liver, brain, or bones. Metastasis may occur early or late in the disease progression, although typically metastasis occurs once the cancerous growth reaches a size of about 20 mm. Metastasis is achieved by cells breaking away from the parental mass and entering either the bloodstream or the lymphatic system.
Genetic inheritance appears to play a role in about 5–10% of breast cancer patients. Mutations in the BRCA1, BRCA2, and p53 tumor suppressor genes have been observed to confer high risks of breast and ovarian cancers. BRCA1 mutations are present at between 1 in 300 to 1 in 800 females. In the BRCA1 gene, over 200 different mutations have been discovered to date. The mutations observed are not localized to a single region, further complicating genetic analysis. Greater than 80% of the observed mutations result in a truncated form of the BRCA1 protein. Individuals with familial hereditary BRCA1 possess one normal and one mutant form of the gene, and are therefore much more likely to develop breast cancer. It is estimated that women with a hereditary BRCA1 mutation are about 76% likely to develop breast cancer by 70 years of age.
BRCA2 has been identified on chromosome 13q through linkage analysis of 15 breast cancer families that did not demonstrate BRCA1 linked breast cancer. Unlike BRCA1 mutations, BRCA2 does not substantially elevate the risk of ovarian cancers. The BRCA2 gene encodes a protein of 3,418 amino acids, many of which are acidic or basic. Most mutations observed involve base deletions that alter the reading frame, and result in a premature truncation of the protein. BRCA1 and BRCA2 account for about 45% of familial inherited breast cancers each, leaving 10% for one or more additional genes. Interestingly, all male breast cancers appear to be due to mutations in the BRCA2 gene.
Mutations found in breast tumor p53 genes are commonly single base pair changes which result in variants with increased cellular half lives. Altered p53 proteins have been observed in 20–25% of breast cancers.
1.3 Deficiencies in the Prior Art
There are relatively few tumor suppressor genes whose mutations have been shown to correlate with the presence of cancerous cells. Those that have been characterized typically have many possible types and positions of mutations, complicating genetic analyses and the prediction of cancer predisposition. Therefore, what is lacking in the prior art is the identification and characterization of novel tumor suppressor genes, and identification of the role of the proteins encoded by such genes in cancer diagnosis and treatment. Such genes, in combination with improved genetic testing, and improved correlation of specific genetic mutations with particular cancer susceptibility are needed to facilitate early and effective treatment of these proliferative diseases.